Ion interaction at the pore of Lc-type Ca²⁺ channel is sufficient to mediate depolarization-induced exocytosis

The coupling of voltage-gated Ca²⁺ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca²⁺ with La³⁺ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd²⁺, nifedipine, or verapamil inhibited depolarization-evoked secretion in La³⁺, indicating specific binding of La³⁺ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La³⁺ > Ce³⁺ > Pr³⁺, consistent with a size-selective binding interface of trivalent cations at the channel pore. La³⁺ inward currents were not detected and the highly sensitive La³⁺/fura-2 imaging assay (∼1 pm) detected no La³⁺ entry, cytosolic La³⁺ build-up, or alterations in cytosolic Ca ². These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca²⁺ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca ²⁺ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca²⁺ sensor protein in the process of neuroendocrine secretion.